Antimicrobial chemokines (AMCs) are understudied members of the family of host defense peptides, which together play an essential role in protecting a wide variety of organisms from bacterial infection. Understanding how AMCs recognize and bind to bacterial targets is key to understanding the basic mechanisms of the innate immune system, as well as designing new antimicrobial therapies that build on millions of years of co-evolution between pathogens and the innate immune system. This application tests the hypothesis that different bacteria are not uniformly affected by AMCs, and identifying bacterial proteins that modulate AMC susceptibility will uncover processes that could provide novel targets for antimicrobial therapy. Since differences in binding to AMCs could be a critical determinant of bacterial susceptibility, we have developed a flow cytometry based assay to measure AMC binding to bacterial cells. Aim 1 involves utilizing this assay to screen thousands of Yersinia transposon mutants to identify those with high AMC binding phenotypes compared to wild type bacteria. Genes associated with lipopolysaccharide biosynthesis appear to play a major role in AMC avoidance and resistance, and Aim 2 involves a detailed characterization of the contributions of these genes to AMC avoidance in both Y. pseudotuberculosis and Y. pestis. Aim 3 tests whether increases in AMC binding due to specific mutations correlate with increased sensitivity to killing by AMCs, as well as by other host defense peptides. Understanding how AMCs and other host defense peptides recognize and bind bacteria, as well as how bacteria evade the antimicrobial activity of host defense peptides, could lead to novel drug targets that enhance the innate immune system. The therapeutic use of AMCs to fight infections may be significantly enhanced by simultaneously targeting processes such as lipopolysaccharide biosynthesis that are required for resistance to these peptides. PUBLIC HEALTH RELEVANCE: This project involves determining how bacterial pathogens resist the body's innate immune defenses. These defenses include a family of peptides called antimicrobial chemokines. These peptides can kill bacteria directly, in addition to their other roles in the immune system. New treatments that interfere with bacterial defense mechanisms could render pathogens less able to cause disease.